Method of propagating microorganisms

Abstract

A method of propagating the Haemophilus gallinarum organism utilizing an aqueous growth medium which produces greater than 10 8 colony forming units per milliliter. The product in this concentration is useful as an immunologic agent effective against infectious coryza in fowl.

We claim: 
     
       1. A medium for propagating Haemophilus gallinarum comprising assimilable sources of carbon and nitrogen in water, the medium further containing as essential ingredients at least the equivalent of sodium ions provided by 5.0 grams per liter of sodium chloride, and reduced nicotinamide adenine dinucleotide in an amount of at least 0.006 grams, per liter, the medium being characterized further by the absence of serum and meat infusion. 
     
     
       2. A medium as defined in claim 1 wherein the sodium ions are present in an amount equivalent to that provided by addition of from 0.5 to 1.0 percent by weight of sodium chloride per liter of medium, and the reduced nicotinamide adenine dinucleotide is present in an amount of about 0.05 grams per liter of medium. 
     
     
       3. A medium as defined in claim 2 containing, per liter of medium, 10 grams polypeptone; ten grams peptone; 3 grams beef extract powder; 0.05 grams nicotinamide; 0.05 grams para-aminobenzoic acid; 1 gram soluble starch; 0.5 grams dextrose; and 2.3 grams phosphate buffer containing thiamine. 
     
     
       4. A process of propagating Haemophilus gallinarum comprising incubating Haemophilus gallinarum in a medium comprised of assimilable sources of nitrogen and carbon in water, the medium further containing sodium ions and reduced nicotinamide adenine dinucleotide, the incubation taking place under an atmosphere comprised of 5 to 15 percent carbon dioxide in air until a product containing at least 10 8  CFU/ml is obtained. 
     
     
       5. The process of claim 4 wherein the atmosphere is 10 percent carbon dioxide and the incubation is for a period of 18 hours at 37°C. 
     
     
       6. A method of producing a bacterin effective against infectious coryza in fowl comprising propagating Haemophilus gallinarum in a medium comprising assimilable sources of nitrogen and carbon in water, the medium being characterized by the absence of serum or meat infusion and further containing sodium ions and reduced nicotinamide adenine dinucleotide, until a product containing at least 10 8  CFU/ml is obtained, and then inactivating the product. 
     
     
       7. The method of claim 6 wherein the product is inactivated by addition of merthiolate. 
     
     
       8. The method of claim 6 wherein the product is inactivated by addition of formalin. 
     
     
       9. The method of claim 6 wherein the bacterin is adjuvanted with aluminum hydroxide gel. 
     
     
       10. The method of claim 6 wherein the bacterin is adjuvanted with Freund's incomplete adjuvant.